The present invention relates to recombinant methods of producing fusion proteins using E. coli proteins as carrier proteins for producing soluble fusion proteins.
A major benefit resulting from the advent of recombinant DNA technology has been the large scale production of proteins of medical or industrial importance. The simplest and most inexpensive means available for obtaining large amounts of proteins by recombinant DNA technology is by expression of protein genes in bacteria (Georgiou and Valax, 1996). The efficient synthesis of heterologous proteins in the bacterium Escherichia coli has now become routine. However, when high expression levels are achieved, recombinant proteins are frequently expressed in E. coli as insoluble protein aggregates termed "inclusion bodies." Although initial purification of inclusion body material is relatively simple, the protein must be subsequently refolded into an active form, which is typically a cumbersome trial-and-error process (Georgiou and Valax, 1996). Thus, it is much more desirable to express the recombinant protein in soluble form.
A strategy to avoid inclusion body formation is to fuse the protein of interest (i.e. the target protein) to a protein known to be expressed at substantial levels in soluble form in E. coli (i.e. the carrier protein). The most widely used carrier protein for the purpose of solubilization is thioredoxin from E. coli (LaVallie et al., 1993). A fusion protein system using thioredoxin for solubilization of target proteins is now being marketed by Invitrogen Corporation (Carlsbad, Calif.). However, despite being touted for its ability to solubilize proteins in a fusion protein, thioredoxin does not always lead to formation of a fusion protein which is soluble at the normal E. coli growth temperature of 37.degree. C. LaVallie et al. (1993) used thioredoxin as a carrier protein to express 11 human and murine cytokines. Of the 11 proteins, only 4 were expressed in soluble form as thioredoxin fusions at 37.degree. C. The non-soluble fusion proteins with thioredoxin could be expressed in soluble form by reducing the growth temperature for expression to as low as 15.degree. C. Several problems with the use of thioredoxin fusions for protein solubilization for any protein are apparent. For example, having to reduce the expression temperature to as low as 15.degree. C. may give unacceptable low rates of protein expression and slow growth rates. Also, due to the small size of thioredoxin (11.7 kilodaltons), fusions with larger proteins may not be soluble; that is, thioredoxin may not be large enough to compensate for the insolubility of a large protein.
Two other E. coli fusion protein systems are widely used: fusions with E. coli maltose-binding protein (Guan et al., 1988), which is 40 kilodaltons in size, and fusions with Schistosoma japonicum glutathione S-transferase (Smith and Johnson, 1988), 26 kilodaltons in size. Both of these systems were developed with the objective of enabling an affinity purification of the fusion protein to be carried out. Both systems tend to give soluble fusion proteins but fail to do so approximately 25% of the time (New England Biolabs Tech Data Sheet, 1992; Smith and Johnson, 1988). Thus, thioredoxin fusions appear to be more soluble than either maltose-binding protein fusions or glutathione S-transferase fusions. An E. coli fusion protein system which could be reliably produced in a soluble form would be desirable.